Method of preparing pterin compounds



Patented July 19, 1949 METHOD OF PREPARING PTERIN COMPOUNDS liecnard Do-uh, Detroit, and Louis L. vJB'ani-liiui',

GrossePointe Woods, Mich assignorsto Parke, Davis & Company, Detroit, Mish -a corporation of :Michigan No Drawing. Application September Eli; are,

Serial No. 697,158

This invention relates to p'terin compounds and a process for: preparing'thevsame. More particw larly, the invention relates to the preparation of 1-(+) -N- [4- (E (2 amino-4ehydroXy-6-pteridyl) methyl]amino)benzoyll 'glutamic acid of the'f'o'rmula,

NH? came-GENE mil-cashmere OOH I N= H OOH 1-(+) -N- [4- [('2 amino 4 -hydrox -.6-'pteridylimethyllamino)benzoyll glutamic acid is an antianemia factor'which can he obtained by hydrolytic or 'enzy'matic degradation of many naturalprodu'cts. In nature, this acid occurs in chemical combination withvaryingnumbers oi'1-(+) glutamic acid residues. For example; the con11ugated product obtained from yeast or liver tissue contains six additional 1=-(+') -glutamic acid residues while that obtained by fermentation contains an additional tWo'l-(+)'-"g'fll1tamic acid residues. In the past the 'free acid has been produced by the autolysis o f liver-tissue and also by the errzymati'c hydrolysis of the conjugated product present in yeast. However, these two methods-oi obtaining this compound are unsatisfactory from a commercial standpoint because" of the small amounts of the conjugated product's present in the starting materials and also because ofithe tedious purification processes which must b'e employed to obtain material suitable for therapeutic use. Recently twomethods of chemical synthesis of this acid have been proposed seeScience, 103, 66'7-9 (1946) The first method consists in reacting equal molar amounts of 2,4,5-tri'amino-6- hydroxypyrimidine, p-aminobenzoyl- 1- -glu-' tamic acid and 2,3-dibromoproplonaldehyde in .11 Claims. (01. cot -251') the presence or acetate "bufier. The second method involves reacting 2,'3-'dibromopropioria1- dehyde with pyridine and "then condensing the resulting product with 12;4=,5 triamino-G-hydroxypyrimidine and potassium iodide to obtain N-[ (2- amino 4-hydrox-y-6 -=pteridyl') methyl] pyridinium iodide; This pteri'ne compound is then reacted with p=aminobenzoyl l-(+ glutamic acid and sodiumimethoxide in ethylene glycol at" C. to produce a material containing the substituted g'lutami'c :acid.

We: have-discovered a method of synthesizing this valuable therapeuticfcoinpound which comprises rea'cting approximately equivalent amounts of an u-halo-ac'rolei-n, l -p-'aminobenzoylglutamic acid and 2;4;5-'triamino-6-hydroxypyrimidine at a pH between about 14' and 8.5 in a reaction medium consisting of water or an aqueous water miscible organic solvent such as dioxane, methanol,iethanol isopropannland acetone. Although theprocess canbee-fie'cted by the simultaneous reaction of the three starting materials, we'prefeniirom'thexstandpoint10f yields, to carry it out in 'a' stepwise fashion. This is accom-' plished by first-condensing the vlit-halo-acrolein with the p-aminobenzoylglutamio acid and then reacting theresulting product with 2,4;5-triamino- 6-hydroxypy-rimidinei Thislatter reaction product which is dihydro-l i-i )'-N-i[ i'-( ['(2-amino-4- hydroxyfi-pteridyb'methylil amino) benzoyl] glutamic acid may either be simultaneously or subsequently oxidized to the desired 1-(+)'-N [4- ([(2 -amino- 4 hydroxy 6 -pteridy-l) methyl=] amino) benzoyll glutamic acid.

The transformations which take place in our process probably can be diagrammatically represented as follows:

Oxidation where X is C1 or Br.

The pH may be controlled throughout the process by the use of a bufier such assodiumacetate, potassium acetate, sodium bicarbonate, disodium hydrogen phosphate and the like. Alternatively, the pH of the initial react-ionmi-xture may be adjusted to a value between 4 and 8.5 and, if necessary, readjusted at the start of the second step of the process. In general, it is not necessary to readjust the pH of the mixture before carrying out the oxidation step ofthe process as the yields are about the same" whether the oxidation is carried out simultaneously with the second step or subsequently as a separate and third step in the process.

The first step of. the process which appears to involve the Lei-addition of the 1-(+) -p-aminobenzoyl-glutamic acid -to the e-halo-acrolein is preferably carried outv in'aqueousldioxane under slightly acidic conditions; e. g.,. at a pH between about 4 and 5, and at a temperature below about 50 C. Under'these conditions'the condensation proceeds quite rapidly toproduc'e a high yield of the intermediate condensation product. This condensation product which probably is a 1-(+) p (p halo B formylethyllaminobenzoylglutamic acidof the formula,

H COOH where X has the same significance as given above, is not isolated but is used without further purification in the next step of the process.

The cyclization or second step of the process is preferably carried out under acidic conditions but at a slightly higher pH than that used in Step I. The pH of the reaction mixture during this step of the'process has a great influence on the color and yield of the final product. At a pH between about 4 and 5 the reaction product contains a dark reddish-brown colored impurity which makes it more difficult to purify the final oxidation product. However, we have found that the formation of this impurity can be eliminated to a large extent by carrying this reaction out at a pH slightly less than '7 but greater than about 5.5.

The yellow colored dihydropteri compoundwhich is produced under these latter conditions yields on oxidation a light colored and readily pu'rifiabie final product.

The exact location of thetwo hydrogenatoms in the dihydropterin compound is not known with certainty but it appears as if they are located either in the 5-6 ond'7-8'positions. However, since both of these dihydro-pterins, or a mixture of the two, yield the desired-pterin compound on oxidation the exact structure of this intermediate product does not influence theyield of the final product. pterin product is not isolated but is converted by Oxidation directly to the desired pterin compound. This oxidation can be carried out at the same time as the cyclization step by vigorous stirring or aeration of the reaction. mixtura ihe'temperature during this simultaneous cyclization and OOH oxidation is kept below about 50 C., preferably at about room temperature, and the pH of the reaction mixture maintained within the limits set forth above. In general, this combination reaction requires about fifteen to twenty-five hours for completion due to the slow rate of the cycliza- Moreover; this intermediate dihydro- Iefiected in the following manner. .material is dissolved in 0.2 N sodium hydroxide tion step.

Alternatively, the cyclization step of the process may be carried out in an inert atmosphere such as nitrogen or methane and then the dihydropterin product present in the reaction mixture oxidized to the corresponding pterin. This two-step reaction is effected in. the following manner: An equivalent amount of solid 2,4,5-triamino-s-hydroxypyrimidine or a solution of the same is added to the reaction mixture obtained from Step I of the process and the resultant mix ture stirred or agitated in the absence of air for about ten to fifteen hours. The reaction mixture containing the dihydro-pterin is then aerated with oxygen or air for about four to six hours to convert the dihydro-ptcrin to the desired pterin compound. The pH and temperature during both of these steps are maintained within the same limits as when carrying out this transformation by simultaneous cyclization and oxidation.

The invention is illustrated by the following examples.

Example 1 2.66 g. of p-aminobenzoyl-l(+)-glutamic acid and 2 g. of sodium acetate are dissolved in cc. of warm water and the solution cooled to room temperature. 1.35 g. of e-bromo-acrolein (Ber. 31, 1385(1898)) is added to the p-a-minobenzoyl- 1(+) -glutamic acid solution with vigorous shaking and the mixture allowed to stand for twenty minutes. At the end of this time the pH of the solution was about 4.5. A solution of 1.4 g. or" 2,4,5-triamino-6-hydroxypyrimidine in 200 cc. of water is prepared in a separate container and then added with vigorous stirring to the solution containing the crude addition product of a-bromoacrolein and p-aminobenZoyl-1(+) -glutamic acid. A fine chocolate colored precipitate starts to separate out of the acidic reaction mixture (pl-I about 4) soon after mixing. The mixture is stirred overnight, the pH adjusted to 3 by the addition of dilute hydrochloric acid and the brownish colored precipitate collected by filtration. After drying in vacuo at room temperature the crude product weighs 2.65 g. It contains 12% ;,by weightof the desired 1-(+) -N- [4-( (Z-amino- 4 hydroxy 6 pteridyl) methyllamino)benzoyllglutamic acid as shown by microbiological assay.

After standing for about two days a second crop of the crude product separates from the reaction mixture filtrate. It is collected and dried as before. This material which Weighs 0.5 g. contains 18.7% of the desired product.

The total yield of the crude product is 3.15 g. while the percentage yield of pure product based on the amount of starting materials used is 9.3%.

The purification of the crude product can be The crude to to C. The precipitate whichseparates is collected, dissolved 0 1 sodium nydroxide ate i usted 5 H co ec e and cr stalliz d: hotzwa a Th pure ;L-.( :-N.- 4 [lg amino-lhydroxy 6-pteridy1).methy1-l minokbenzovll utami d gradually darkens with charring and without melting on heating. {Depending on the. rate of heating, decomposition-sets in, as evidenced by darkening of .color,at around 250 ---C.,and a gradual charring as the temperature is raised to 360 0. Its ultra viQI tabsQrPt Qn spect u v.111 0.005 N sodium ;hydroxld e.jex ibigts athree absorption maxima very close-to the awavej-lengthszzfifi mu, 282 mu and 365 mu, with Bits of approximately 542, 53iand 194 respectively, and absorption minima very close to' the wave lengths 235 mu, 268 mu and 333 mu, with in water but it is quite insoluble inanhydrous -nbutanol (less than 0:005 per 330.)

The product exercises a growth-stimulating effect on Lactobacillus casei and an ,antianemia vitamin effect in chicks suffering from a deficiency of this acid. ln human therapy it is useful in treatment of non-pernicious .anemias.

Example 2 a o dium acetatea d.:z t isa i-re min n yl-i +l--s utamie a id-a e lssq d in of is lled er h le Mark t n -the so ution with nitrogen. :Niroeen 5 bu b ed thrwsh h s u n a d lee-m .e-brcmoacm einlsadded with stirring. Theqnirixt-nreis-stirred for ten minutes and the resultant solution added to a solution .of 1.55,g...of 2,4;fietriaminoefi hydroxypyrimidine in 200 cc. of water; During this additionand the subsequent Jreaction the mixture :is covered with an atmosphere oi. nitrogen. A heavy chocolate colored precipitate separates from the solution soon I after. mixing -.-.the.- tworeactants. The reaction vessel is-stoppei ed 'and shaken tor forty eight hours. Therea-ctionmixture is removedirom the reaction vessel and aerated ior four hours byblowing through the solution to oxidize the :dihydro. 1-f( -N.-: [1416i (Zaamino- 4-hydroxy 6 pteridyl(methyllaminoibenzoyll lutamic acid. At the end of the :aerationithe pH of the solution is about 5. The precipitate :of the crude product is collected, washed withhold-water and dried in vacuo at room temperature. -The 1 weight of the crude productlis 2; 7 g. which contains 11.1% of 'the desired "'1 -'(-i-)-=N-'-['.4-'(['(2'- amino 4 hydroxy-6-pteridyl)methyllamino)- benzoyll glutamic acid as shown byiinicmbiological assay on LdctobaciZlus casez; If the pure material isdesired"itmaybe ob tained from this crude product by thepurification method described inExample 1.

is dissolved in 10 cc. of :warm -;a'queous dioxane, the solution cooled-and neutralized:topHi'I with 1 N sodium hydroxide: solution.- 0.-14.:g,rof a-bromo-acrolein in 1.5 cc; of'dioxane i's added with vigorous shaking, the mixturevallowed i170 stand for 10 minutes and then diluted toa volum-e of 20 cc. with 50% aqueous dioxane. Thisdiluted solution'is added with stirring to a solutionof 0.15 g. of 2,4,5-triamino-6 hydroxypyrimidine in 20 cc. of water, the reaction:vesselstopperednnd shaken overnight. The reactionvesselds opened and the mixture. aerated for three lioursby bubbling air through the Solution. The pH :of the solution which at this time is 4.8 is adjusted to 3.5 and the mixture diluted-to 100 cc.'rwith distilled water. The brown colored crudeproduct is collected, washed with cold water and dried in vacuo at room temperature. The weight of this material which contains 13.3% lmicro biological assay) of 1-(+)-N-[4-([(2-amino-4- hydroxy 6 pteridyDmethyllamino)benzoyll glutamic acid is 0.17 g. If desired the crude product may be purified as described in lilxeample 1.

- Example 4 1.35 g. of a-bromo-acrolein is addedto 2:66:. of p-amino-benzoyl--1(+)-glutamic acid dissolved in 30 cc. of dilute methanol at 2491C. The temperature of the reaction mixture-rises 4 C. and a yellow color develops immediately. After the mixture has stood for five or six minutes-3 g. of sodium acetate is added andthe solution covered with methane. After. standing for one hour 1.4 g. of 2,4,5-triamino-6-hydr0XyDyrimidine in 500 cc. of 50% methanolis added and the: mixture allowed to stand under methaneovernight.

The reaction mixture is aerated for three hours by bubbling air through the solution andthe I crude product collected by filtration and dried.

The crude material weighs 1 g. and contains 8.5% (by microbiological assay) of pure .1-(-+ N- [4- (2-amino-4-hydroxy-S-pteridyllmethyll amino) benzoyl] glutamic acid. The pure acid may be obtained from thiscrude material by following the purification procedure..describedgin Example 1. a Example .5

2.66 g. of p-aminobenzoyl-1 )-glutaniic acid is dissolved in 10 m1. of 50% aqueous'dioxane and the pH of the solution adjusted to 7.5 by the addition of 1 N sodium hydroxide solution. 0.9 g. of a-chloro-acrolein in a small amount of. dioxane is added and the mixture stirred for one and a half hours. The'pI-I of the solution isagainadjusted to 6.5 and then added to a Summoner-0.15 g. of 2,4,5-triamino-fi hydroxypyrimidine' M1 20 cc. of water. The'reaction mixture is stirred o'v'er night, the pH adjusted to 3.5 and the crude brown colored product collected. After drying the product weighs about 1 g. and contains about (by microbiological assay) of pure 1-(+) -N-'[4- ([(2-amino 4 hydroxy 6 pteridyDmethyllamino)benzoyl] glutamic acid. The pure acid may be obtained from the crude material by the purification procedure of Example 1.

What We claim as our invention is:

1. Process for obtaining 1-(+) -N-[4-([(2- amino 4 hydroxy-G-pteridyl) -methylla.mino) benzoyl] glutamic acid of formula,

which comprises reacting an a-halmacrolein of the class consisting of a-ChlOl'O and a-bromo acrolein, peamino-benzoyl 1- (+)-glutamic acid and 2,4,5-triamino-6-hydroxy pyrimidine below about 50 C. at a pH between about 4 and 8.5 in a reaction medium of the class consisting of water and aqueous water-miscible organic solvents and oxidizing the dihydro-l(+)-N-[4- ([2 amino 4 hydroxy 6 pteridyDmethyllamino benzoyll glutamic acid so formed with gaseous oxygen, thereby obtaining 1-(+)-[4- ([(2 amino 4 hydroxy-G-pteridyl) -methy1lamino benzoyll glutamic acid.

2. Process for obtaining (1-(+)-N-[4-([(2- amino 4 hydroxy-G-pteridyl)methyllamino) benzoyl] glutamic acid of formula,

which comprises condensing an a-halo-acrolein of the class consisting of a-ChlOlO and a-bromoacrolein with p-aminobenzoyl-1(+)-glutamic acid, reacting the condensation product with 2,4,5-triamino-6-hydr0Xy-pyrimidine and oxidizing the dihydro-1-(+) -N- [4- (2-amino-4-hydroxy 6 pteridyl) methyl] amino) benzoyl] glutamic acid so formed with gaseous oxygen, thereby obtaining 1- [4- E (2-amino-4-hydroxy-G-pteridyl) -methyll amino) benzoyl] glutamic acid, said process being carried out in a reaction medium of the class consisting of water and aqueous water miscible organic solvents at a pH between about 4 and 8.5 and at a temperatime below about 50 C.

3. Process for obtaining (1-(+)-N-[4-'([(2- amino 4 hydroxy-G-pteridyl)methyll amino) a benzoyl] glutamic acid of formula,

which comprises condensing a-bromo-acrolein with p-aminobenzoyl-1-(+)-glutamic acid, reacting the condensation product with 2,4,5-triamino-6-hydroxypyrimidine and oxidizing the dihydro 1 N-[4-([(2-amino-4-hydroxy-6- pteridyl) methyl] amino) benzoyl] glutamic acid so formed with gaseous oxygen, thereby obtaining 1 [4-(E(2-amino-4-hydroxy-6-pteridyl) -methyllamino)benzoyl] glutamic acid, said process being carried out in a reaction medium of the class consisting of water and aqueous water miscible organic solvents at a pH between 201300132 4 and 8.5 and at a temperature below about 8 4. Process ,for obtaining (1-(+)-N-[4-([(2- amino 4 hydroxy-S-pteridyl)methyllamino) benzoyl] glutamic acid of formula,

which'comprises condensing an oc-hfiIO-BLIOISHI of the classconsisting of a-ChIOio and a-blOIIlO- acrolein with p-aminobenzoyl 1(+) glutamic acid'at a pH between about 4 and 5, reacting the condensation product with 2,4,5-triamino-6-hydroxypyrimidine at a pH slightly less than '7 but greater than about 5.5 and oxidizing the dihydro- 1-(+) -N- [4- (2-amino-'i-hydroxy-G-pteridyl) methyll'aminolbenzoyll glutamic acid so formed with gaseous oxygen, thereby obtaining 14+)- [4 (2-amino-4-hydroXy-6-pteridyl) -methyl] amino) benzoyl] glutamic acid, said process being carried out in a reaction medium of the class consisting of water and aqueous water-miscible organic solvents at a temperature below about 50 C.

5. Process for obtaining (1-(+)-N-[4-([(2- amino 4 ydroxy-G-pteridyl)methyllamino) benzoyl]v glutamic acid of formula,

which comprises condensing an a-halo-acrolein of the class consisting of a-OhlOlO- and a-bromoacrolein with p-aminobenzoyl 1(+) -glutamic acid at a pH between about 4 and 5, reacting the condensation product with ,4,5-triamino-6-hydroxypyrimidine at a pH slightly less than 7 but greater than about 5.5 and simultaneously oxidizing the dihydro-l- -N- [4- (2-amino-4-hydroxy 6-pteridyl)methyl]amino) benzoyl] glutamic acid so formed with gaseous oxygen, thereby obtaining 1-(+) -[4-([(2-amin0-4-hy- 4 5 droxy-G-pteridyl) -methyllamino) benzoyl] glutamic acid, said process being carried out in a reaction medium of the class consisting of water and aqueous water-miscible organic solvents at a temperature below about C.

6. Process for obtaining (1-(+)-N-[4-([(2- amino 4 hydroxy-G-pteridyl)methyllamino) benzoyl] glutamic acid of formula,

which comprises condensing an a-halo-acrolein of the class consisting of a- 6h101'0 and a-bromoacrolein with p-aminobenzoyl-1(+)-glutamic acid at a pH between about 4 and 5, reacting the condensation product with 2,4,5-triamino-6-hydroxypyrimidine in an inert atmosphere at a pH slightly less-than 7 but greater than about 5.5 and'subsequently oxidizing the dihydro-l-(+)- N- [4-( (2-amino-4-hydroxy-B-pteridyl) methyl] amino)benzoyl] glutamic acid so formed with gaseous' oxygen, thereby obtaining 1-(+)-[4- (2 amino 4 hydroxy G-pteridyDmethyllamino)benzoyll glutamic acid, said process being carried out in a reaction medium of the class consisting of water and aqueous water-miscible 23%3310 solvents at a temperature below about 7.111 a Process for obtaining 1-(+) -N-E4-(E(2- 9 amino 4 hydroxy-G-pteridyl) methyllamino) benzoyl] glutamic acid of formula,

the step which comprises condensing an a-haloacrolein of the class consisting of oc-GhlOlO- and a-bromo-acrolein with p-aminobenzoyl-1(+) glutamic acid in a reaction medium of the class consisting of water and aqueous water-miscible organic solvents at a pH between about 4 and 8.5 and at a temperature below about 50 C. to obtain a 1- p- (p-halo- B-formylethyl) ami nobenzoyl glutamic acid of probable formula,

X OOH where X is a member of the class consisting of Cl and Br.

8. In a process for obtaining 1- N- [4-( (2- amino 4 hydroxy-G-pteridyl)methyllamino)- benzoyl] glutamic acid of formula,

N( i-N= H COOH the step which comprises condensing an a-haloacrolein of the class consisting of a-ChlOlO- and a-bromo-acrolein with p-aminobenzoyl-1(+) glutamic acid in aqueous dioxane at a, pH between about 4 and 5 and at a temperature below about 50 C. to obtain a 1-(+) p-(p-halo-p-formylethyl) aminobenzoyl glutamic acid of probable formula,

the steps which comprise reacting a 1-(+)-p- (B halo p-formylethyl)aminobenzoylglutamic acid of the class consisting of the p-chloroand fi-bromo-derivatives of said acid which has the probable formula,

COOH

where X is a member of the class consisting of Cl and Br, with 2,4,5-triamino-G-hydroxypyrimidine and oxidizing the dihydro-1-(+) N-[4- ([(2 amino 4 hydroxy 6-pteridyl)methyl]- amino) benzoyl] glutamic acid so formed with gaseous oxygen, thereby obtaining 1-(+)-[4- ([(2 amino 4 hydroxy G-pteridyDmethyllamino)benzoyl] glutamic acid, said steps being carried out in a reaction medium of the class consisting of water and aqueous water-miscible organic solvents at a pH between about 4 and 8.5 and at a temperature below about 50 C.

10. In a process for obtaining 1-(+)-N-[4- ([(2 amino-4-hydroxy-6-pteridyl) methyllamino) benzoyl] glutamic acid of formula,

the steps which comprise reacting a 1-( +)-p- (,8 halo ,B-formylethyl)aminobenzoylglutamic acid of the class consisting of the ,B-chloroand p-bromo-derivatives of said acid which has the probable formula,

OOH

where X is a member of the class consisting of Cl and Br, with 2,4,5-triamino-6-hydroxypyrimidine at a pH slightly less than 7 but greater than about 5.5 and simultaneously oxidizing the d'ihydro 1- N- [4- (2-amino-4-hydiroxy-6- pteridyl) methyl] amino) benzoyll glutamic acid so formed with gaseous oxygen, thereby obtaining 1 [4-([(2-amino-4-hydroxy-6-pteridyDmethyllamino)benzoyl] glutamic acid, said steps being carried out in a reaction medium of the class consisting of water and aqueous watermiscible organic solvents at a temperature below about 50 C.

11. In a process for obtaining 1-(+)-N-[4- [(2 amino-4-hydroxy-G-pteridyl) methyllamino)benzoyl] glutamic acid of formula,

the steps which comprise reacting a 1-(+)-p- 6 halo ,B-formylethyl)aminobenzoylglutamic acid of the class consisting of the B-chloroand p-bromo-derivatives of said acid which has the probable formula,

0 0 I I t i-c11-0HzNHOc-NH-cH-omcmooo11 H X GOOH No references cited. 

